Profiling The Developmental Changes Of Phosphorylated Proteome In Eimeria Tenella Life Cycle

Eimeria tenella is the main pathogen with a complex life cycle that causes coccidiosis in chickens.It’s metabolism pathways and protective antigens are changed with its different developmental stages.However,little is known about the developmental regulation and transformation mechanisms of its life cycle.Studying the proteomics,the phosphoproteomics and the interactions between the kinome and the phospho-proteins in different stages are indispensable in revealing the regulatory mechanisms,understanding the adaptive changing mechanisms on each metabolism pathway and also aiding for drug targets discovery.Firstly,lable-free quantitative proteomics approach was utilized to identify protein expression profiles in E.tenella with different developmental stages.A total of 957 proteins across all the stages were used to construct the weighted gene co-expression network(WGCN)and seven modules were identified.Proteins in red,green,blue and brown modules were screened out to be significantly positively correlated with the E.tenella developmental stages of unsporulated oocysts(USO),sporulated oocysts(SO),sporozoites(S)and second-generation merozoites(M2),respectively.Hub proteins with a high intramodule connectivity were identified for each module.Functional enrichment analyses revealed that some hub proteins in green and blue modules were involved in electron transport chain and oxidative phosphorylation,indicating that energy metabolism is associated with sporulating and sporozoite invasion.And hub proteins in brown modules were engaged in alternative splicing,indicating that many protein isoforms were generated in stage differentiation.Secondly,the protein phosphorylation data was harvested using lable-free phosphoproteomics approach for the E.tenella with four developmental stages.A total of 456,479,and 198 differentially expressed phosphoproteins(DEPPs)were identified in the comparisons of partially sporulated(7h)oocysts vs.unsporulated oocysts,sporulated oocysts vs.partially sporulated(7h)oocysts,and sporozoites vs.sporulated oocysts,respectively.Functional enrichment analyses of DEPPs suggested that they were involved in diverse functions,including cell division,actin cytoskeleton organization,peptide metabolic process,translation,tricarboxylic acid metabolic process and calcium ion binding.Time course sequencing data analysis identified six clusters with different expression patterns on the four developmental stages,each with similar expression pattern related to carbohydrate metabolism,cytoskeleton organization,regulation of exocytosis and calcium ion transport,demonstrating different regulatory profiles across the life cycle of E.tenella.These results revealed significant changes in the abundance of phosphoproteins during E.tenella development,and involved in diverse functions.Thirdly,we applied the Hidden Markov Model(HMM)approach to identify and classify the kinome in E.tenella.In total,96 members in the E.tenella kinase superfamily were identified,and were classified into 11 groups.The results of ortholog inference analysis indicated that there are gene duplication for some specific protein kinases during the course of evolution.Finally,the results of proteomics and phosphoproteomics were applied to construct the dynamic correlation network between E.tenella protein kinases and phosphorylated proteins by Pearson correlation analysis.In the correlation network,several protein kinases and phosphorylated protein pairs were significantly correlated during the development of E.tenella.And multiple protein kinases tend to clump together in cluster and have formed a dense cluster with their related phosphorylated proteins.Our correlation analysis results indicated that protein kinases and phosphoproteins in the dense clusters might take part in the same or similar pathway.In summary,this work is the first research studying on proteomics,phosphoproteomics and kinome in different developmental stages for E.tenella.WGCNA analysis was performed and the weighted gene co-expression network was constructed with the proteomics data.Several hub proteins related to the four developmental stages were identified.Phosphoproteomics analyses revealed significant changes in the abundance of phosphoproteins with diverse functions during E.tenella development.The dynamic correlation network between the protein kinase and the phosphoproteins proteins of E.tenella was also constructed.Our study provides a data foundation for further studying of the regulatory mechanism and pathogenesis of developmental stages of E.tenella.